Based on the fact that the salivary gland undergoes continuous replacement, it is hypothesized that all cell types that are found in the adult salivary gland are reformed by stem or progenitor cells, and that these cells can be used to reform salivary tissues upon in vivo transplantation. Studies were initiated this year that are aimed at determining whether it is possible to isolate a clonogenic, self-renewing cell from salivary glands, and if there is a common stem cell, able to reform all cell types within a salivary gland, or a hierarchy of stem or progenitor cells that are more committed to one particular phenotype or another. Other studies are under way to devise methods of in vivo transplantation, a critical assay for the identification of a stem cell, and also for potential clinical applications. As a first approximation, techniques that have recently been established to isolate stem cells from pancreatic ductal and acinar elements are being used. Murine and human salivary glands are treated enzymatically to produce a cell suspension and plated in serum-free medium containing keratinocyte growth factor or hepatocyte growth medium. Cells derived from these procedures are being characterized for expression of various marker proteins, including cytokeratins 7, 8, 18, and 19 to examine the general salivary and epithelial nature of the cells, and with multiple antibodies that can be used to distinguish acinar and ductal cellular phenotypes. (amylase and claudin). While it is important to characterize in vitro differentiation, the gold standard for stem cell studies is in vivo transplantation. Male murine cells and human cells are being transplanted as into female immunocompromised mice. The donor origin of the tissue that is generated can be determined by FISH (Y chromosome in mouse into mouse transplants) and by in situ hybridization with human specific alu sequences (human into mouse transplants). To date, cells have been combined with Matrigel and placed into immunocompromised animals either subcutaneously. The resulting transplants have been analyzed histologically, and duct-like structures have been identified. Current studies are aimed at improving the growth of clones derived from murine and human salivary glands (which is exceedingly slow), and to develop better in vivo transplantation assays to support the formation of acinar tissues.